Assessment of sample freezing as a preservation technique for analysing the molecular composition of dissolved organic matter in aquatic systems

Dissolved organic matter (DOM) is widely studied in environmental and biogeochemical sciences, but is susceptible to chemical and biological degradation during sample transport and storage. Samples taken in remote regions, aboard ships, or in large numbers need to be preserved for later analysis without changing DOM composition. Here we compare high-resolution mass spectra of solid phase extractable DOM before and after freezing at −20 °C. We found that freezing increases compositional dissimilarity in DOM by between 0 to 18.2% (median = 2.7% across 7 sites) when comparing replicates that were frozen versus unfrozen, i.e., processed immediately after sampling, as compared with differences between unfrozen replicates. The effects of freezing primarily consisted of a poorer detection limit, but were smaller than other sample preparation and analysis steps, such as solid phase extraction and variable ionisation efficiency. Freezing samples for either 21 or 95 days led to similar and only slight changes in DOM composition, albeit with more variation for the latter. Therefore, we conclude that sample freezing on these time scales should not impede scientific study of aquatic DOM and can be used where it makes logistical sense, such as for large spatial surveys or study of archived samples.


Introduction
Characterising dissolved organic matter (DOM) composition is critical in environmental and biogeochemical studies, but technical and logistical limitations oen restrict the number of samples that can be immediately processed. 1,2 For instance, sampling sites can be far from laboratories, 3 and intensive sampling campaigns are oen condensed over a short period with limited time for sample preparation. 4 As additional constraints, ultra-high-resolution mass spectrometry (UHRMS), such as Orbitrap or Fourier-transform ion cyclotron mass spectrometry, which is the gold standard method to measure DOM composition, 5 oen requires a time-consuming solid phase extraction (SPE) step that is traditionally performed as soon as possible aer sampling to avoid sample degradation. 6 Therefore, without a way to preserve collected samples, only a limited number can be analysed, which limits our understanding of global biogeochemical cycles. To allow DOM characterisation in remote areas, on archived samples, and to embrace the realisation of high throughput environmental studies, long-term storage solutions are needed.
Sample poisoning and freezing are among the most used techniques to preserve DOM and prevent biological and photochemical degradation, 7 but no studies have tested how they affect DOM composition measured using UHRMS. Adding inorganic chemicals (e.g. HgCl 2 , NaCl) into samples is costefficient and can be performed anywhere under any conditions. 7 However, most substances that prevent biological activity are harmful to humans and the environment, 8 and cause difficulties for sample transport because of hazardous goods regulations. In the case of nontoxic chemicals (e.g. NaCl), downstream complications arise such as the requirement of several washing steps to remove the excess of inorganic salts prior to processing samples with UHRMS. 6 Moreover, introducing a compound inside a sample increases the likelihood of sample modication or contamination. Preserving samples at sub-zero temperatures is a simple and cost-effective alternative that is achievable in all areas with electricity. Once frozen, samples can be stored for months without further effort. There are also many laboratories with decades of archived frozen samples that might be appropriate for compositional analysis using modern techniques.
Evidence from studies measuring dissolved organic carbon (DOC) in both marine and freshwater samples aer freezing are encouraging, with DOC concentrations remaining unchanged for several months aer initial freezing. [9][10][11] Although freezing samples changes DOM composition measured using optical properties, 9,11,12 these effects may not be relevant to UHRMS. A major drawback of freezing water is the formation of crystals which alter the three-dimensional (3D) structure of molecules. 13 Differences in 3D structure can modify DOM optical properties, 14 but 3D structures are not resolved in UHRMS. While freezing may also modify DOM by rupturing microbial cells and releasing intracellular compounds, these cells are typically removed during pre-ltering of samples through 0.20, 0.45, or 0.70 mm, which are operational denitions for DOM. 6 Therefore, it is likely that the molecular formulae in DOM are conserved aer freezing, even though the 3D congurations have been altered. Overall, the inconveniences of sample poisoning likely outweigh their benets, whereas freezing samples may be more suitable for preserving samples before UHRMS measurements.
The initial DOM composition of a sample might further affect its resilience to freezing. Natural DOM comprises diverse compound groups with various structures, ranging from large aromatic (e.g. lignin, tannins) to small aliphatic (e.g. lipids, peptides) 15 compounds. Solely based on chemical processes, molecular formulas that are more reactive may be more affected by freezing than compounds decaying at slower rates. 16 Conversely, compounds that are more prone to occulation may be more susceptible to loss during freezing. Therefore, environments dominated by highly unsaturated and phenolic compounds, such as boreal lakes, 16 might be affected differently by freezing than environments containing higher proportions of aliphatic compounds, such as small clearwater streams, 17 marine waters, 18 or aerosols. 19,20 Understanding how compositional responses to freezing vary among sample types is therefore essential to guide preservation efforts.
In this study, we studied the effects of freezing on UHRMS DOM composition (mass spectra and common peak metrics) in two separate experiments. The rst experiment investigated fresh and coastal marine waters in Sweden before and aer 21 days of storage at −20°C following a standard protocol for marine studies. 21 In the second experiment, we investigated freshwaters in the UK during a longer freezing period (95 days) following a protocol designed for a global scale study. We only tested the effect of freezing at −20°C, as it is the typical temperature used in previous studies. 9,11 Our results indicate that freezing had a very minor effect on UHRMS results, and so we propose freezing as a sensible option to preserve samples where necessary.

Experimental design
Samples were collected in Sweden and the UK (Fig. 1) to compare primarily the effects of shorter and longer freezing times, respectively. On 18 May 2022, surface water (0.5 m depth) was collected from three Swedish sites draining predominantly forested land: two lakes, Strömsvattnet (sampled from open water) and Ned Färingen (sampled in a reed bed), and a small drainage ditch. A coastal marine sample was obtained from Tjärno Marine Station located on the west coast of Sweden on 19 May 2022, off the end of a nearby jetty. We hereaer refer to these sites as Swedish lake (Strömsvattnet), reed bed (Ned Färingen), drainage ditch, and marine (Tjärno Marine Station). During July 2020, we collected samples from three sites in the UK: the River Mel (sampled on 17 July 2020), Coe Fen (sampled on 17 July 2020), and Loch Ken (sampled on 23 July 2020). The River Mel is a small, clear-water chalk stream located near the city of Cambridge. Coe Fen is a wetland inside the city of Cambridge that dries during the summer and is relled during winter and aer rainfall events. Loch Ken is a brown-water Scottish loch surrounded by forest plantations and agriculture in an area with relatively low population density. We hereaer refer to these sites as chalk stream (River Mel), fen (Coe Fen) and British lake (Scottish loch). All the UK samples were collected from surface waters (0.5 m depth) near the centre of the sampling site.
At each site we ltered six replicates into pre-combusted (4 h at 550°C) glassware. In Sweden, we passed the water through pre-combusted 25 mm GF/F grade lters (Whatman, UK) loaded into a Swinnex syringe capsule (MilliporeSigma, USA), which is a standard protocol for DOM analyses of marine and freshwater samples. 6 In the UK, we used pre-assembled 25 mm syringe lters with 0.2 mm cellulose acetate membranes (Sartorius, UK). Pre-assembled lters can be more practical in citizen science or sampling networks involving many different eld teams, especially in remote eld conditions where there may be limited access to laboratory facilities for pre-combustion of lter papers. At each site in Sweden and the UK, a new lter was used for each replicate sample, and the rst 30 mL ltrate was discarded. Three replicates were acidied to a pH of 2 using 37% HCl, and prepared for SPE, and three replicates were transferred to a −20°C freezer where they were stored for 21 (Sweden) or 95 days (UK). Aer the freezing period, samples were thawed in a fridge (ca. 4°C) for 24 hours (Sweden) or at room temperature for 3 hours (UK), acidied to a pH of 2 using 37% HCl, and prepared for SPE. During thawing, one vial broke from the River Mel, and the water was transferred to a clean glass beaker for analysis. At each site, we collected an additional sample as described above into a pre-combusted 40 mL glass vial, which was kept in the dark at ca. 4°C for up to one week prior to quantication of DOC concentration.

Solid phase extraction
Agilent PPL cartridges (100 mg styrene-divinylbenzene) 6 were pre-conditioned using a 3 mL MeOH ush followed by a 4 hour MeOH soak and a 3 mL acidied Milli-Q™ (0.1% HCl) ush. Samples were loaded into 60 mL syringes and le to gravity feed the cartridges. A second 3 mL acidied Milli-Q™ ush was used to remove any salts before eluting the cartridges with 2 mL MeOH, approximately 1.8 mL of which was recovered into precombusted 4 mL amber vials. The vials were stored upright at −20°C until analysis. On each occasion, SPE was also performed on a Milli-Q™ blank. The volume of samples extracted is presented in ESI Table 1. †

Mass spectrometry analysis
For each replicate in Sweden, 1.2 mL eluate was pipetted into a Milli-Q™ rinsed Eppendorf™ tube and dried in a Speedvac for 3 hours at 30°C. Dry samples were then re-dissolved in 100 mL ACN + 0.1% FA, and brought back into solution by vortexing then sonicating for 5 minutes. The supernatant (80 mL) was then pipetted into a pre-combusted autosampler vial (1.5 mL with 300 mL inserts). 10 mL of sample was injected into the LC-MS for freshwater samples and 30 mL for marine samples.
For the UK replicates, the amount of eluate to be dried was determined based on an expected 60% extraction efficiency and with a target concentration of 100 ppm. Dried eluates were redissolved in 120 mL ACN + 0.1% FA containing internal standards of capsaicin (100 mL of 1000 ppm stock), adenosine-5monophosphate (10 mL of ppm 1000 stock), raffinose (100 mL of 1000 ppm stock), carbenoxolone disodium (10 mL of 1000 ppm stock), and glycyrrhizic acid (10 mL of 1000 ppm stock), and the supernatant (100 mL) was transferred to an autosampler vial by Hamilton syringe which was washed between samples with methanol. 80 mL of each sample was injected into the LC-MS.
The MS (Orbitrap LTQ Velos, Thermo Fisher, Germany) was optimised in negative ion mode using direct infusion of a 20 ppm SRNOM solution in 50% methanol, tuning to maximise the intensity of the ion at 369.11911. Analytes were separated by reversed phase chromatography with a Kinetix Polar C18 column (100 × 2.1 mm, 2.6 mm, Phenomenex, Torrance, USA) and two mobile phases (A and B). Mobile phase A was 0.1% FA in LCMS grade H 2 O (i.e. 100 mL H 2 O + 100 mL FA). Mobile phase B was 0.1% FA in 80 : 20 (v/v) LCMS grade ACN : water. A second pump provided a counter gradient post-column (Han et al., 2021), balancing the solvent composition to a constant 40% ACN. The mobile phases in the second pump were spiked with hippuric and fusidic acid (10 mL of 1000 ppm stock each per 100 mL) for the Swedish samples and hippuric, fusidic and cyclohexyl succinic acid (10 mL of 1000 ppm stock each per 100 mL) for the UK samples. The spiked compounds are expected to be absent in DOM samples and allow continuous calibration of mass at various m/z values. 22 Compounds were separated over a 10 minute gradient: 0% B for 0.5 minutes, up to 100% B at 6 minutes, isocratic till 6.5 minutes, down to 0% B at 7 minutes, 3 minute equilibration at 0.22 mL min −1 . Aer mixing with the counter gradient mobile phase, 10% of the total ow was diverted to the electrospray ionisation (ESI) source for analysis by UHRMS. The ESI was set to −3 kV, 100°C, and the Orbitrap was set to collect data at a resolution setting of 60 000 (the second highest setting), to increase the total number of transients collected. The data were exported as a single integrated mass list.

DOC concentration
DOC samples were analysed on a Shimadzu TOC-V (Sweden) or TOC-L (UK) analyser (Shimadzu, Japan) and quantied as nonpurgeable organic carbon (NPOC). Measurements were made in triplicate. For Swedish samples, pure water (Milli-Q) blanks and an ethylenediaminetetraacetic acid (EDTA, 8 mg C per L) standard were included throughout the analysis run. For UK samples, deionised (DI) water blanks and a 10 mg C per L potassium hydrogen phthalate standard were used throughout for QA/QC. For both studies, the measured values were within 5% of the expected values.
Before sampling, we also determined the concentration of water required to rinse the cellulose acetate lters used in the UK so that they did not release any DOC. Briey, we ushed DI water in increments of 10 mL from 0 to 50 mL through a cellulose acetate syringe lter. Then, we passed an additional 25 mL of DI water through the lter and collected the ltrate. We measured DOC concentration in the ltrate and in the DI water. Passing 20 mL of DI water through the lter was enough to reduce the DOC to the original concentration of the DI water (mean ± standard error: ltered DI = −0.003 ± 0.052 mg C per L; original DI = 0.008 ± 0.031 mg C per L, N = 3).

Data analysis
A formula assignment routine was written in MATLAB (version R2021b, Mathworks, USA), which is available along with raw sample data in the ESI. † Formulas were assigned between masses 150-850 Da, with the following constraints: Noise was removed using the Kendrick mass defect slice method, 23 and high molecular weight doubly charged interference peaks were removed from consideration before assignment. 22 A mass error of 1 ppm and 1.75 ppm was allowed for the Swedish dataset and UK dataset respectively, aer evaluating assignment errors aer calibration ( Fig. SI1 and 2 †). All peaks that were not assigned a molecular formula were removed from the dataset. Remaining peak intensities were normalised to sum 1 × 10 6 for each sample, and normalised intensities were used both for averaging (for comparison before and aer freezing), and as the basis of Bray-Curtis dissimilarity testing.
We tested the effect of freezing on the number of peaks, the intensity weighted average O/C (O/C wa ), H/C (H/C wa ), and massto-charge (m/z wa ) ratios using linear models. The linear models were tted to each metric using the function lm in R 4.1.1, with predictor of site and treatment and their interaction, i.e. to test if freezing effects varied with DOM composition. Furthermore, for each site, individual peak intensities before and aer freezing were compared using paired t-tests. To compare overall composition of samples, the Bray-Curtis dissimilarity between each sample was computed and used for visualising the variability between treatments with a principal coordinate analysis (PCoA).

Short-term (21 day) storage of Swedish fresh and marine waters
Freezing samples from the Swedish sites for three weeks did not have a statistically signicant effect on any of the peak metrics under investigation ( Table 1). The mean and standard deviation of the intensity weighted peak metrics among replicate samples within frozen or unfrozen (i.e., processed immediately aer sampling) treatments were more similar than between treatments (frozen minus unfrozen samples, Table 1). Furthermore, freezing samples did not increase the dissimilarity between replicates, irrespective of the sites considered ( Fig. 2 and Table  2). The effect size, measured as the difference in Bray-Curtis dissimilarities between unfrozen samples only versus between frozen and unfrozen samples, ranged from an average of 0.0 to 2.7% (Table 2).
We observed statistically signicant changes in the intensity of some individual peaks (Fig. 3). We found a total of 8255, 8149, 8092, and 7059 peaks were present in at least one of the replicates from the Swedish lake, reed bed, drainage ditch, and marine sites, respectively (Fig. 3). Of those peaks, 3, 6, 14, and 9% (with a sum intensity respectively corresponding to, 8, 7, 6, and 12% of the total sum intensity of each sample) were signicantly (paired t-test, p < 0.05) affected by freezing in the Table 1 High resolution mass spectrometry peak metrics showing average number of peaks (peaks) and mean intensity weighted average of oxygen to carbon (O/C wa ), hydrogen to carbon (H/C wa ) and mass to charge (m/z wa ) ratios. N = 3 for each sample set (unfrozen or frozen), and N = 9 for the comparison between frozen and unfrozen samples. SD = standard deviation. There was no statistically significant difference between frozen and unfrozen samples (p > 0.05)

Sample
Peak ( Swedish lake, reed bed, drainage ditch, and marine sites, respectively. Molecular formulae exhibiting altered intensity varied depending on the sampling site. In the drainage ditch and marine sample, compounds with a H/C > 1 and O/C of 0.2-0.6, oen assigned to carboxyl-rich alicyclic molecules (CRAMs), decreased in relative intensity aer freezing (Fig. 3). In the drainage ditch, molecular formulae with a H/C < 1 and O/C > 0.4, oen associated with tannins, decreased in relative intensity. Other sites had very minor changes without clear patterns in van Krevelen space (Fig. 3).

Long-term (95 days) storage of UK freshwaters
Freezing samples for 95 days had a statistically signicant effect on intensity weighted peak metrics for the chalk stream and British lake (Table 3). For the chalk stream, the number of peaks detected and the m/z wa ratio decreased by 45% and 6%, on average, respectively (Table 3). For the British lake, the O/C wa , and the m/z wa ratio decreased by 2%, and 4%, on average, respectively, whereas the H/C wa increased by 7%, on average ( Table 3). The Bray-Curtis dissimilarity between replicates were smaller before compared with aer freezing samples for the chalk stream and British lake (Fig. 4). The difference in peak intensity between replicates was exacerbated by one replicate for the chalk stream, for which the vial broke during processing, and that was 30% dissimilar to the other replicates (F-CS-3 in Fig. 4). The effect size, measured as the difference in Bray-Curtis dissimilarities between unfrozen samples and the Bray-Curtis dissimilarities between frozen and unfrozen samples, ranged from an average of 5.0% to 18.2% (Table 4). More pronounced changes were found at the level of individual peak abundances in the second study compared with the rst (Fig. 3 and 5). We found a total of 2164, 2839, and 2049 peaks were present in at least one of the replicates from the chalk stream, British lake, and fen sites, respectively (Fig. 3). Of those peaks 28, 29% and 11% (respectively 11, 45, and 13% of the intensity) were signicantly (paired t-test, p < 0.05) affected by freezing for chalk stream, British lake, and the fen sites respectively. For the chalk stream, most changes occurred for compounds related to CRAMs (H/C > 1 and O/C of 0.2-0.6), with these mostly decreasing in relative intensity (Fig. 5) like in the Swedish drainage ditch and marine sample (Fig. 2). For the British lake, compounds related to aliphatics (H/C > 1. 5 (Fig. 5). For the fen, few clear patterns emerged in van Krevelen space (Fig. 5).

Technical reproducibility
In our dataset, the SRNOM peak metrics varied the least on each occasion, as these were analytical replicates from a single vial. On average, we found more peaks, a higher H/C wa ratio, a lower O/C wa ratio, and a lower m/z wa in the SRNOM processed with the Swedish sites (Tables 1 and 5) than the SRNOM processed with the UK sites (Tables 3 and 5). However, the differences in peak metrics between the SRNOM remained small and did not affect the conclusion of this study. The intensity weighted average metrics for the SRNOM standard were also compared with recent data from an international ring trial. 5 Because the current data were obtained by liquid chromatography separation with counter gradient, which increases the detection of compounds with a high O/C ratio, 24 and not by direct infusion as used in the ring trial, some differences were expected. Compared with the direct infusion of the ring trial, we found that high O/C and low H/C peaks were slightly over-represented in our analysis, along with higher mass compounds (especially for the UK sites), but that our instrument and method remained suitable for analysing DOM (Table 5).

Effect of freezing on DOM composition
In this study, we have demonstrated that freezing samples does not have major consequences on overall peak metrics (H/C, O/C,  ), and those with a statistically significant relative decrease and increase after freezing are superimposed in red and blue (filled circles) as a gradient of % change, respectively. In the right panel, the logarithm of average intensity (I norm ) of unfrozen and frozen samples are shown on x-and y-axes, respectively. Unchanged peaks are shown as grey circles, and red/blue colours signify decreased/increased peaks, respectively, corresponding to the van Krevelen diagram. The numbers in the mass spectra are the number of peaks in each group. m/z, or peak number) or on the intensity of individual peaks in northern temperate waters. Specically, freezing samples had smaller effects on DOM composition than reports of other sample preparation techniques routinely used in DOM sample analysis by mass spectrometry. Our results showed that compositional changes induced by freezing were usually much less than 10% (Tables 2 and 4). Therefore, like SPE 25 and choice of ionisation technique, 26,27 sample freezing does inuence sample composition. However, the effects of freezing were lower than those of SPE, which has been shown to increase the difference between technical replicates by ca. 15% for samples processed with a styrene-divinylbenzene polymer (PPL) sorbent. 15 Furthermore, more than 40% of the carbon is usually lost during the SPE process, irrespective of freezing, indicating that a large fraction of the sample is not recovered. 25,28 Ionisation techniques are also hugely important in dictating which peaks are observed, 26,29 and electrospray source geometry and settings may be one of the main reasons for different results between laboratories. 5 A recent estimate indicates that only 33% of the standard Suwannee River fulvic acid is efficiently ionised by ESI in negative mode. 27 Overall, the combination of SPE and ESI provides the analyst with a very specic, but nevertheless important analytical window through which the sample is viewed. The addition of the minor sample freeze-thaw effect on top of these major biases should be considered a 'necessary evil' in cases where, for logistical or archiving reasons, the sample needs to be frozen. Furthermore, unlike for SPE or sample ionisation, it is possible to both quantify and characterise the effects of freezing, rendering data interpretation more manageable.
The effects of freezing are highly consistent and reproducible. Out of 21 frozen samples, only one replicate was clearly Table 3 High resolution mass spectrometry peak metrics showing number of peaks (peaks) and the intensity weighted average of oxygen to carbon (O/C wa ), hydrogen to carbon (H/C wa ) and mass to charge (m/z wa ). Bolded numbers indicate a statistically significant difference (p < 0.05) after freezing. N = 3 for each sample set (unfrozen or frozen), and N = 9 for the comparison between frozen and unfrozen sample. SD = standard deviation  different from the other replicates. That replicate (F-CS-3, Fig. 4) was broken during thawing, and it is likely that unnoticed contamination occurred during transfer to new glassware. The potential for contamination emphasises the need for sample replication, which is oen not undertaken in UHRMS DOM studies. 5 Our results suggest very high reproducibility of the results between replicates both before and aer freezing, and in most cases between unfrozen and frozen samples. In the few cases where there was a statistically signicant change aer freezing (e.g. for the chalk stream), freezing primarily decreased the peak number and average mass, and not the O/C or H/C metrics (Tables 1 and 3).  ), and those with a statistically significant relative decrease and increase after freezing are superimposed in red and blue (filled circles) as a gradient of % change, respectively. In the right panel, the logarithm of average intensity (I norm ) of unfrozen and frozen samples are shown on x-and y-axes, respectively. Unchanged peaks are shown as grey circles, and red/blue colours signify decreased/increased peaks, respectively, corresponding to the van Krevelen diagram. The numbers in the mass spectra are the number of peaks in each group.
Compositional changes underlying shis in peak intensity reected the sampling environment. The reed bed (Sweden) and British lake (UK) exhibited an increase in aliphatic and CRAM formulas, while more aromatic formulas decreased ( Fig. 3 and  5). Conversely, the more terrestrial drainage ditch (Sweden) and fen (UK) sites, exhibited an increase in tannin-like compounds and decrease in more aliphatic compounds, essentially the opposite effect. The marine site (Sweden), Swedish lake (Sweden) and chalk stream (UK) had no consistent patterns in van Krevelen space related to freezing ( Fig. 3 and 5), but as stated, peak numbers decreased considerably for the chalk stream (Table 3 and Fig. 5). These effects suggest that the intrinsic properties of compounds, such as 3D structures and intermolecular bonds, and compound chemistry related to site (bio) geochemistry does lead to freezing effects, and that these effects are consistent and reproducible between biogeochemically similar sites.
Our study may also have implications for freezing of methanol extracts aer SPE. This practice is widespread but may similarly affect DOM composition. It has previously been determined that, at room temperature, DOM carboxylic acid groups can form methyl esters with solvent methanol. 30 This reaction is much slower at −20°C, but presumably not halted entirely over years of storage. Extracts could be stored aer drying, 31 but this practice is uncommon and the effects of drying remain unknown. Furthermore, drying extracts or samples under eld conditions is oen impractical due to equipment requirements. Future efforts should attempt to determine the effects of long-term storage at −20°C on methanol extracts if the need to re-measure samples becomes relevant, particularly if this is considered a better alternative to freezing of site water.

Comparison between the two case studies
We observed a smaller effect of freezing aer 21 days than aer 95 days. Besides the freezing time, the type of lter, the volume of sample extracted, the amount of carbon injected into the HPLC-Orbitrap, and the sampling sites differed between the two experiments. Because both types of lters were ushed and no leaching of carbon was observed for the cellulose acetate lters, it is unlikely that the type of lter contributed to the differences between the two experiments. Passing more water through the cartridge only increased the number of peaks detected in the UK sites (Table S1 †) and is therefore unlikely to explain the observed effects of freezing between the Swedish and UK studies (Tables 2 and 4). However, future studies should aim to keep extraction volumes as consistent as possible to avoid potential differences during analysis. Sites in Sweden and the UK differed in their concentrations of inorganic ions, which might interfere with the extraction efficiency of DOM. Furthermore, two of the three UK sites had a higher H/C ratio than three of the four Swedish sites (Tables 1 and 3), indicating a broad difference in DOM chemistry between the two sets. Therefore, our results suggest that the larger effect of freezing in the UK sites is likely caused by the longer freezing time, differences in water chemistry, or differences in extraction volumes. Further work would be required to fully deconvolute site (e.g. salt content, DOC concentration) and storage time effects.

Conclusions
In this study, we showed that freezing can change DOM composition, but that any changes are relatively small (typically < 10%) and can be both quantied and characterised. Importantly, freezing never impeded distinction of DOM from different study sites. A good practice for future UHRMS studies of frozen DOM would be to quantify and characterise biases with a set of contrasting reference materials, in addition to ensuring sample replication. Furthermore, we found that the duration of freezing between 21 and 96 days did not have strong effects on DOM composition. These results suggest that samples may remain representative of their original composition even aer many more months, thereby offering the potential to analyse long-term archives. Nonetheless, further studies evaluating the effects of freezing over very long time scales (e.g. years) would be benecial. Our results also raise new questions about processing frozen methanol extracts, as is routinely done 4,6 during UHRMS. Some of the effects we observed in water samples might also occur in methanol extracts, in addition to methylation of carboxylic acids to form methyl esters. Overall, we conclude that freezing samples can produce a small but measurable bias that may be acceptable for large-scale and/or remote environmental studies.

Conflicts of interest
There are no conicts to declare.